Integrated heater and magnetic separator

ABSTRACT

An apparatus for providing thermal and magnetic energy to a receptacle containing a reaction mixture and a magnetic retention member. The apparatus can also control heating of a reaction mixture, and bring about a separation of magnetic particles from the reaction mixture. The reaction mixture typically comprises polynucleotides from a biological sample that are being brought into a PCR-ready form.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/482,572, filed Apr. 7, 2017 and scheduled to issue as U.S. Pat. No.10,139,012 on Nov. 27, 2018, which is continuation of U.S. patentapplication Ser. No. 12/178,586, filed Jul. 23, 2008 and issued as U.S.Pat. No. 9,618,139 on Apr. 11, 2017, which is a continuation-in-part ofU.S. patent application Ser. No. 12/173,023, filed Jul. 14, 2008 andissued as U.S. Pat. No. 8,133,671 on Mar. 13, 2012, and U.S. patentapplication Ser. No. 12/218,498, filed Jul. 14, 2008 and issued as U.S.Pat. No. 9,186,677 on Nov. 17, 2015, both of which claim benefit ofpriority to U.S. Provisional Patent Application No. 60/959,437, filedJul. 13, 2007, all of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

The technology described herein generally relates to an apparatus forproviding thermal and magnetic energy to a receptacle containing areaction mixture and a magnetic retention member. The technology moreparticularly relates to an apparatus for controlled heating of areaction mixture, and for bringing about a separation of magneticparticles from the reaction mixture. The reaction mixture typicallycomprises polynucleotides from a biological sample that are beingbrought into a PCR-ready form.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, many diagnostic analyses can only bedone with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations—often just one in any given urban area. This means that mosthospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss or mishandling. Second, the equipment inquestion is typically not available ‘on-demand’ but instead runs inbatches, thereby delaying the processing time for many samples becausethey must wait for a machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using PCR to amplify a vector of interest. Onceamplified, the presence of a nucleotide of interest from the sampleneeds to be determined unambiguously. Preparing samples for PCR iscurrently a time-consuming and labor intensive step, though not onerequiring specialist skills, and could usefully be automated. Bycontrast, steps such as PCR and nucleotide detection have customarilyonly been within the compass of specially trained individuals havingaccess to specialist equipment.

Sample preparation is labor intensive in part because most samples mustbe heated at one or more stages, and in part because targetpolynucleotides are typically captured by some kind of retention memberwhich must then be effectively isolated from the surrounding milieu.Thus, even where various liquid transfer operations can be optimized,and even automated, there is still a need for controlled application ofheat, and efficient capture of extracted polynucleotides in situ.

The discussion of the background herein is included to explain thecontext of the inventions described herein. This is not to be taken asan admission that any of the material referred to was published, known,or part of the common general knowledge as at the priority date of anyof the claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

An apparatus for separating magnetic particles, comprising: one or moremagnets affixed to a supporting member; a motorized mechanism configuredto move the supporting member in such a manner that the one or moremagnets move backwards and forwards along a fixed axis, and during atleast a portion of the motion, the one or more magnets maintain closeproximity to one or more receptacles which contain the magneticparticles; and control circuitry to control the motorized mechanism.

An integrated separator and heater, comprising: a heater assembly,wherein the heater assembly comprises a plurality of independentlycontrollable heater units, each of which is configured to accept and toheat one of a plurality of process tubes; one or more magnets affixed toa supporting member; a motorized mechanism configured to move thesupporting member in such a manner that the one or more magnets movebackwards and forwards along a fixed axis, and during at least a portionof the motion the one or more magnets maintain close proximity to one ormore of the process tubes in the heater assembly, wherein the one ormore process tubes contain magnetic particles; and control circuitry tocontrol the motorized mechanism and to control heating of the heaterunits.

A diagnostic apparatus comprising the integrated separator and heater asdescribed herein.

A method of controllably heating a plurality of process tubes, eachcontaining a solution of reagents and biological samples, whereinconditions in each of the process tubes may be individually tailored.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of an automated apparatus configured to carryout sample preparation using a heater and separator as described herein.

FIGS. 2A and 2B show an exemplary embodiment of a reagent holder, inperspective view (FIG. 2A), and underside view (FIG. 2B).

FIG. 3 shows a heater unit in perspective and cross-sectional view.

FIG. 4 shows perspective views of the rack of reagent holders and sampletubes of FIG. 5, in conjunction with a heater unit.

FIG. 5 shows perspective views of an exemplary rack for samples andreagent holders.

FIG. 6 shows an integrated heater and separator unit in cross-sectionalview.

FIG. 7 shows an exemplary heater/separator.

Like reference numerals in the various drawings indicate like elements.

DETAILED DESCRIPTION

The heater and separator described herein are typically configured foruse in a method and apparatus for carrying out sample preparation onbiological samples in parallel, with or without PCR and detection on theprepared samples, and preferably with high throughput.

Apparatus Overview

A schematic overview of an apparatus 981 for carrying out automatedsample preparation on multiple samples in parallel, according to stepsexemplified elsewhere herein, is shown in FIG. 1. The geometricarrangement of the components of system 981 is exemplary and notintended to be limiting. The apparatus may additionally comprise (notshown in FIG. 1) a microfluidic cartridge, in a receiving bay, andconfigured to carry out a diagnostic test on the sample, such as bydetecting presence of an amplified polynucleotide in the cartridge. Suchadditional features are also described in U.S. patent application Ser.No. 12/173,023, filed on Jul. 14, 2008 (and entitled “IntegratedApparatus for Performing Nucleic Acid Extraction and Diagnostic Testingon Multiple Biological Samples”, in the name of Williams, et al.).

A processor 980, such as a microprocessor, is configured to controlfunctions of various components of the system as shown, and is therebyin communication with each such component requiring control. It is to beunderstood that many such control functions can optionally be carriedout manually, and not under control of the processor. Furthermore, theorder in which the various functions are described, in the following, isnot limiting upon the order in which the processor executes instructionswhen the apparatus is operating. Thus, processor 980 can be configuredto receive data about a sample to be analyzed, e.g., from a samplereader 990, which may be a barcode reader, an optical character reader,or an RFID scanner (radio frequency tag reader).

Processor 980 can be configured to accept user instructions from aninput device 984, where such instructions may include instructions tostart analyzing the sample, and choices of operating conditions.Processor 980 can be also configured to communicate with a display 982,so that, for example, information about an analysis is transmitted tothe display and thereby communicated to a user of the system. Suchinformation includes but is not limited to: the current status of theapparatus; progress of PCR thermocycling; and a warning message in caseof malfunction of either system or cartridge. Additionally, processor980 may transmit one or more questions to be displayed on display 982that prompt a user to provide input in response thereto. Thus, incertain embodiments, input 984 and display 982 are integrated with oneanother. Processor 980 can be optionally further configured to transmitresults of an analysis to an output device 986 such as a printer, avisual display, a display that utilizes a holographic projection, or aspeaker, or a combination thereof. Processor 980 can be still furtheroptionally connected via a communication interface such as a networkinterface to a computer network 988.

Processor 980 can be further configured to control various aspects ofsample preparation and diagnosis, as follows in overview. In FIG. 1, theapparatus 981 is configured to operate in conjunction with acomplementary rack 800. Apparatus 981 may be capable of receivingmultiple racks, such as 1, 2, 3, 4, or 6 racks.

Embodiments of rack 800 are further described in U.S. patent applicationSer. No. 12/______, filed by ExpressMail on Jul. 14, 2008 (and entitled“Integrated Apparatus for Performing Nucleic Acid Extraction andDiagnostic Testing on Multiple Biological Samples”, in the name ofWilliams, et al), and 12/______, filed on even date herewith, andentitled “Rack For Sample Tubes And Reagent Holders”, in the name ofDuffy, et al., both of which are incorporated herein by reference intheir entireties. A rack 800 is itself configured to receive a number ofbiological samples 996 in a form suitable for work-up and diagnosticanalysis, and a number of holders 804—as further described herein, suchas in connection with FIGS. 10A, 10B, that are equipped with variousreagents, pipette tips and receptacles. The rack is configured so that,during sample work-up, samples are processed in the respective holders,the processing including being subjected, individually, to heating andcooling via heater assembly 977.

The heating functions of the heater assembly 977 can be controlled bythe processor 980. Heater assembly 977 operates in conjunction with aseparator 978, such as a magnetic separator, that also can be controlledby processor 980 to move into and out of close proximity to one or moreprocessing chambers associated with the holders 804, wherein particlessuch as magnetic particles are present. Assembly 977 and separator 978are further described herein.

Liquid dispenser 976, which similarly can be controlled by processor980, is configured to carry out various suck and dispense operations onrespective sample, fluids and reagents in the holders 804, to achieveextraction of nucleic acid from the samples. Liquid dispenser 976 cancarry out such operations on multiple holders simultaneously.

Sample reader 990 is configured to transmit identifying indicia aboutthe sample, and in some instances the holder, to processor 980. In someembodiments a sample reader is attached to the liquid dispenser and canthereby read indicia about a sample above which the liquid dispenser issituated. In other embodiments the sample reader is not attached to theliquid dispenser and is independently movable, under control of theprocessor. Liquid dispenser 976 is also configured to take aliquots offluid containing nucleic acid extracted from one or more samples anddirect them to storage area 974, which may be a cooler. Area 974contains, for example, a PCR tube corresponding to each sample.

Embodiments of the apparatus shown in outline in FIG. 1, as with otherexemplary embodiments described herein, are advantageous because they donot require locations within the apparatus suitably configured forstorage of reagents. Therefore, the apparatus in FIG. 1 isself-contained and operates in conjunction with holders 804, wherein theholders are pre-packaged with reagents, such as in locations within itdedicated to reagent storage.

The apparatus of FIG. 1 may be configured to carry out operation in asingle location, such as a laboratory setting, or may be portable sothat they can accompany, e.g., a physician, or other healthcareprofessional, who may visit patients at different locations. Theapparatus is typically provided with a power-cord so that they canaccept AC power from a mains supply or generator. The apparatus may alsobe configured to operate by using one or more batteries and therefore isalso typically equipped with a battery recharging system, and variouswarning devices that alert a user if battery power is becoming too lowto reliably initiate or complete a diagnostic analysis.

The apparatus of FIG. 1 may further be configured, in other embodiments,for multiplexed sample analysis and/or analysis of multiple batches ofsamples, where, e.g., a single rack holds a single batch of samples.Each component shown in FIG. 1 may therefore be present as many times asthere are batches of samples, though the various components may beconfigured in a common housing.

The apparatuses as described herein find application to analyzing anynucleic acid containing sample for any purpose, including but notlimited to genetic testing, and clinical testing for various infectiousdiseases in humans.

The apparatus herein can be configured to run on a laboratory benchtop,or similar environment, and can test approximately 45 samples per hourwhen run continuously throughout a normal working day. Results fromindividual raw samples are typically available in less than 1 hour.

Heater Assembly

A cross-sectional view of a heater unit of an exemplary heater assembly1401 is shown in FIG. 3 (right hand panel). The heater assemblycomprises one or more independently controllable heater units, each ofwhich comprises a heat block. In certain embodiments there are 2, 3, 4,5, 6, 8, 10, 12, 16, 20, 24, 25, 30, 32, 36, 40, 48, or 50 heater unitsin a heater assembly. Still other numbers of heater units, such as anynumber between 6 and 100 are consistent with the description herein. Theone or more heat blocks may be fashioned from a single piece of metal orother material, or may be made separately from one another and mountedindependently of one another or connected to one another in some way.Thus, the term heater assembly connotes a collection of heater units butdoes not require the heater units or their respective heat blocks to beattached directly or indirectly to one another. The heater assembly canbe configured so that each heater unit independently heats each of theone or more process tubes 1402, for example by permitting each of theone or more heat blocks to be independently controllable, as furtherdescribed herein.

In the configuration of FIG. 3, the heater assembly comprises one ormore heat blocks 1403 each of which is configured to align with and todeliver heat to a process tube 1402. Each heat block 1403 can beoptionally secured and connected to the rest of the apparatus using astrip 1408 and one or more screws 1407 or other adhesive device(s). Thissecuring mechanism is not limited to such a configuration.

Although a cross-sectional view of one heat block 1403 is shown in FIG.3, it should be understood that this is consistent with having multipleheat blocks aligned in parallel to one another and such that theirgeometric midpoints all lie on a single linear axis, though it is not solimited in configuration. Thus, the one or more heat blocks may bepositioned at different heights from one another, in groups or,alternately, individually, or may be staggered with respect to oneanother from left to right in FIG. 3 (right hand panel), in groups oralternately, or individually. Additionally, and in other embodiments,the heat blocks are not aligned parallel to one another but are disposedat angles relative to one another, the angles being other than 180°.Furthermore, although the heat block shown in FIG. 3 may be one ofseveral that are identical in size, it is consistent with the technologyherein that one or more heat blocks may be configured to accept and toheat process tubes of different sizes.

The exemplary heat block 1403 in FIG. 3 (right hand panel) is configuredto have an internal cavity that partially surrounds a lower portion ofprocess tube 1402. In the heat block of FIG. 3, the internal cavitysurrounds the lower portion of process tube 1402 on two sides but notthe front side (facing away from magnet 1404) and not the rear side(adjacent to magnet 1404). In other embodiments, heat block 1403 isconfigured to surround the bottom of process tube 1402 on three sides,including the front side. Still other configurations of heat block 1403are possible, consistent with the goals of achieving rapid and uniformheating of the contents of process tube 1402. In certain embodiments,the heat block is shaped to conform closely to the shape of process tube1402 so as to increase the surface area of the heat block that is incontact with the process tube during heating of the process tube. Thus,although exemplary heat block 1403 is shown having a conical,curve-bottomed cavity in which a complementary process tube is seated,other embodiments of heat block 1403 have, for example, a cylindricalcavity with a flat bottom. Still other embodiments of heat block 1403may have a rectilinear internal cavity such as would accommodate acuvette.

Moreover, although heat block 1403 is shown as an L-shape in FIG. 3,which aids in the transmittal of heat from heating element 1501 and insecuring the one or more heat blocks to the rest of the apparatus, itneed not be so, as further described herein. For example, in someembodiments heating element 1501 may be positioned directly underneathprocess tube 1402.

Each heat block 1403 is configured to have a low thermal mass whilestill maintaining high structural integrity and allowing a magnet toslide past the heat blocks and the process tubes with ease. A lowthermal mass is advantageous because it allows heat to be delivered ordissipated rapidly, thus increasing the heating and cooling efficiencyof the apparatus in which the heater assembly is situated. Factors thatcontribute to a low thermal mass include the material from which a heatblock is made, and the shape that it adopts. The heat blocks 1403 cantherefore be made of such materials as aluminum, silver, gold, andcopper, and alloys thereof, but are not so limited.

In one embodiment, the heat block 1403 has a mass of ˜10 grams and isconfigured to heat up liquid samples having volumes between 1.2 ml and10 μl. Heating from room temperature to 65° C. for a 1 ml biologicalsample can be achieved in less than 3 minutes, and 10 μl of an aqueousliquid such as a release buffer up to 85° C. (from 50° C.) in less than2 minutes. The heat block 1403 can cool down to 50° C. from 85° C. inless than 3 minutes. The heat block 1403 can be configured to have atemperature uniformity of 65±4° C. for heating up 1 ml of sample and85±3° C. for heating up 10 μl of release buffer. These ranges aretypical, but the heat block can be suitably scaled to heat other volumesof liquid at rates that are slower and faster than those described. Thisaspect of the technology is one aspect that contributes to achievingrapid nucleic acid extraction of multiple samples by combination ofliquid processing steps, rapid heating for lysis, DNA capture andrelease and magnetic separation, as further described herein andelsewhere, such as U.S. patent application Ser. Nos. 12/172,208 and12/172,214, both of which are incorporated herein by reference.

Not shown in FIG. 3, the heater assembly 1401 can also optionally becontained in an enclosure that surrounds the heat blocks 1403. Theenclosure can be configured to enable sufficient air flow around theprocess tubes and so as not to significantly inhibit rate of cooling.The enclosure can have a gap between it and the heat blocks tofacilitate cooling. The enclosure can be made of plastic, but is not solimited. The enclosure is typically configured to appear aesthetic to auser.

As shown in FIG. 3, the heater assembly 1401 can also comprise one ormore heating elements (e.g., a power resistor) 1501 each of which isconfigured to thermally interface to a heat block 1403 and dissipateheat to it. For example, in one embodiment, a power resistor candissipate up to 25 Watts of power. A power resistor is advantageousbecause it is typically a low-cost alternative to a heating element.Other off-the-shelf electronic components such as power transistors mayalso be used to both sense temperature and heat. Although the heatingelement 1501 is shown placed at the bottom of the heat block 1403, itwould be understood that other configurations are consistent with theassembly described herein: for example, the heating element 1501 mightbe placed at the top or side of each heat block 1403, or directlyunderneath process tube 1402. In other embodiments, the heating elementhas other shapes and is not rectangular in cross section but may becurved, such as spherical or ellipsoidal. Additionally, the heatingelement may be moulded or shaped so that it conforms closely orapproximately to the shape of the bottom of the process tube. Not shownin FIG. 3, the heater assembly can also comprise an interface material(e.g., Berquist q-pad, or thermal grease) between the heating element1501 and the heat block 1403 to enable good thermal contact between theelement and the heat block.

In the embodiment shown in FIG. 3, the heater assembly further comprisesone or more temperature sensors 1502, such as resistive temperaturedetectors, to sense the respective temperatures of each heat block 1403.Although a temperature sensor 1502 is shown placed at the bottom of theheat block 1403, it would be understood that other configurations areconsistent with the assembly described herein: for example, thetemperature sensor might be placed at the top or side of each heat block1403, or closer to the bottom of process tube 1402 but not so close asto impede uniform heating thereof. As shown in the embodiment of FIG. 3,the heater assembly can further comprise an interface material (e.g.,Berquist q-pad) 1503 configured to enable good thermal contact betweenthe sensor 1502 and the heat block 1403, to thereby ensure an accuratereading.

Certain embodiments of the diagnostic or preparatory apparatus hereinhave more than one heater assembly as further described herein. Forexample, a single heater assembly may be configured to independentlyheat 6 or 12 process tubes, and an apparatus may be configured with twoor four such heater assemblies.

Rack

Process tubes 1402 are typically disposed in reagent holders thatthemselves are supported in a rack, as shown in FIG. 4, the combinationof reagent holders and rack ensuring that the process tubes areeffectively located in proximity to the heater units.

The racks for use herein are typically configured to be insertable into,and removable from, a diagnostic or preparatory apparatus as furtherdescribed herein, each of the racks being further configured to receivea plurality of reagent holders, and to receive a plurality of sampletubes, wherein the reagent holders are in one-to-one correspondence withthe sample tubes, and wherein the reagent holders each containsufficient reagents to extract polynucleotides from a sample and placethe polynucleotides into a PCR-ready form. Exemplary reagent holders arefurther described elsewhere herein and also in copending applicationSer. No. 12/______, filed by ExpressMail on Jul. 14, 2008 (and entitled“Reagent Tube, Reagent Holder, and Kits Containing Same”, in the name ofWilson, et al.) and incorporated herein by reference. An exemplaryapparatus is outlined herein, and also described in U.S. patentapplication Ser. No. 12/______, filed by ExpressMail on Jul. 14, 2008(and entitled “Integrated Apparatus for Performing Nucleic AcidExtraction and Diagnostic Testing on Multiple Biological Samples”, inthe name of Williams, et al.), incorporated by reference herein.

Two perspective views of an exemplary rack 800, configured to accept 12sample tubes and 12 corresponding reagent holders, in 12 lanes, areshown in FIG. 5. A lane, as used herein in the context of a rack, is adedicated region of the rack designed to receive a sample tube andcorresponding reagent holder. A perspective view of the same exemplaryrack, in conjunction with a heater unit, as further described herein, isshown in FIG. 4.

A rack may accept 2, 4, 6, 8, 10, 12, 16, or 20 samples such as insample tubes 802, and a corresponding number of holders 804. Thus theembodiment of FIG. 5 configured to receive 12 samples and 12corresponding reagent holders is exemplary.

Magnetic Separator

The disclosure herein further comprises a magnetic separator, configuredto separate magnetic particles, the separator comprising: one or moremagnets affixed to a supporting member; a motorized mechanism configuredto move the supporting member in such a manner that the one or moremagnets move backwards and forwards along a fixed axis, and during atleast a portion of the motion, the one or more magnets maintain closeproximity to one or more receptacles which contain the magneticparticles in solution; and control circuitry to control the motorizedmechanism.

The disclosure herein still further includes an integrated magneticseparator and heater, comprising: a heater assembly, wherein the heaterassembly comprises a plurality of independently controllable heaterunits, each of which is configured to accept and to heat one of aplurality of process tubes; one or more magnets affixed to a supportingmember; a motorized mechanism configured to move the supporting memberin such a manner that the one or more magnets move backwards andforwards along a fixed axis, and during at least a portion of the motionthe one or more magnets maintain close proximity to one or more of theprocess tubes in the heater assembly, wherein the one or more processtubes contain magnetic particles; and control circuitry to control themotorized mechanism and to control heating of the heater units.

Typically, each of the one or more receptacles is a process tube, suchas for carrying out biological reactions. In some embodiments, closeproximity can be defined as a magnet having a face less than 2 mm awayfrom the exterior surface of a process tube without being in contactwith the tube. It can still further be defined to be less than 1 mm awaywithout being in contact with the tube, or between 1 and 2 mm away.

Typically the magnetic particles are microparticles, beads, ormicrospheres capable of binding one or more biomolecules, such aspolynucleotides, and commonly available as retention members. Separatingthe particles, while in solution, typically comprises collecting andconcentrating, or gathering, the particles into one location in theinside of the one or more receptacles.

An exemplary magnetic separator 1400 is shown in FIG. 6, configured tooperate in conjunction with heater assembly 1401. The magnetic separator1400 is configured to move one or more magnets relative to the one ormore process tubes 1402. While the magnet 1404 shown in FIG. 6 is shownas a rectangular block, it is not so limited in shape. Moreover, theconfiguration of FIG. 6 is consistent with either having a single magnetthat extends across all heat blocks 1403 or having multiple magnetsoperating in concert and aligned to span a subset of the heat blocks,for example, aligned collinearly on the supporting member. The magnet1404 can be made of neodymium (e.g., from K &J Magnetics, Inc.) and canhave a magnetic strength of 5,000-15,000 Gauss (Brmnax). The poles ofthe magnets 1404 can be arranged such that one pole faces the heatblocks 1403 and the other faces away from the heat blocks.

Further, in the embodiment shown in FIG. 6, the magnet 1404 is mountedon a supporting member 1505 that can be raised up and down along a fixedaxis using a motorized shaft 1405. The fixed axis can be vertical. Inthe embodiment shown in FIG. 6, a geared arrangement 1406 enables themotor 1601 to be placed perpendicular to the shaft 1405, thereby savingspace in the apparatus in which magnetic separator 1400 is situated. Inother embodiments, the motor is placed underneath shaft 1405. It wouldbe understood that other configurations are consistent with the movementof the magnet relative to the process tubes, including, but not limitedto, moving the magnet from side-to-side, or bringing the magnet downfrom above. The motor can be computer controlled to run at a particularspeed; for example at a rotational speed that leads to vertical motionof the magnet in the range 1-20 mm/s. The magnetic separator can thus beconfigured to move repetitively, e.g., up an down, from side to side, orbackwards and forwards, along the same axis several times. In someembodiments there is more than one shaft that operates under motorizedcontrol. The presence of at least a second shaft has the effect ofmaking the motion of the separator more smooth. In some embodiments, thesupporting member rides on one more guiding members to ensure that thesupporting member does not, for example, tip, twist, or yaw, or undergoother internal motions while moving (other than that of controlledmotion along the axis) and thereby reduce efficacy of the separation.

The supporting member can also be configured to move the magnets betweena first position, situated away from the one or more receptacles, and asecond position situated in close proximity to the one or morereceptacles, and is further configured to move at an amplitude about thesecond position where the amplitude is smaller than a distance betweenthe first position and the second position as measured along the shaft.

Shown in FIGS. 26 and 27, the heater assembly 1401 and the magneticseparator 1400 can be controlled by electronic circuitry such as onprinted circuit board 1409. The electronic circuitry 1409 can beconfigured to cause the heater assembly 1401 to apply heat independentlyto the process tubes 1402 to minimize the cost of heating and sensing.It can also be configured to cause the magnetic separator 1400 to moverepetitively relative to the process tubes 1402. The electroniccircuitry 1409 can be integrated into a single printed circuit board(PCB). During assembly, a plastic guide piece can help maintain certainspacing between individual heat blocks 1403. This design can benefitfrom use of off-the-shelf electronics to control a custom arrangement ofheat blocks 1403.

Not shown in FIGS. 26 and 27, an enclosure can cover the magneticseparator 1400 and the heater assembly 1401 for protection ofsub-assemblies below and aesthetics. The enclosure can also be designedto keep the heat blocks 1403 spaced apart from one another to ensureefficiency of heating and cooling. The magnetic separator and heaterassembly can, alternatively, be enclosed by separate enclosures. The oneor more enclosures can be made of plastic.

Advantageously, the heater assembly and magnetic separator operatetogether to permit successive heating and separation operations to beperformed on liquid materials in the one or more process tubes withouttransporting either the liquid materials or the process tubes todifferent locations to perform either heating or separation. Suchoperation is also advantageous because it means that the functions ofheating and separation which, although independent of one another, areboth utilized in sample preparation, may be performed with a compact andefficient apparatus.

Reagent Holders

Described herein and elsewhere are reagent holders for holding andtransporting reagents for various purposes, in particular samplepreparation in a clinical context, and configured to be received by arack as described herein. The reagent holders also typically provide acontainer in which various reagents can be mixed one with another and/orwith a sample. The holders are also configured for use in an automatedpreparatory apparatus that can carry out sample preparation on samplesin more than one holder simultaneously.

FIGS. 10A and 10B show views of an exemplary holder 804 as furtherdescribed herein. This exemplary holder, as well as others consistentwith the written description herein though not shown as specificembodiments, are now described. Further details of reagent holders canbe found in U.S. patent application Ser. No. 12/______, filed byExpressMail Jul. 14, 2008 in the name of Wilson, et al., and entitled“Reagent Tube, Reagent Holder, and Kits Containing Same”, which isincorporated herein by reference.

The exemplary holder of FIG. 2A comprises a connecting member 510 havingone or more characteristics as follows. Connecting member 510 serves toconnect various components of the holder together. Connecting member 510has an upper side 512 and, opposed to the upper side, an underside 514.

The reagent holder of FIG. 2A is configured to comprise: a process tube520 affixed to the connecting member and having an aperture 522 locatedin the connecting member; at least one socket 530, located in theconnecting member, the socket configured to accept a disposable pipettetip 580; an optional pipette sheath 570 as further described herein; twoor more reagent tubes 540 disposed on the underside of the connectingmember, each of the reagent tubes having an inlet aperture 542 locatedin the connecting member; and one or more receptacles 550, located inthe connecting member, wherein the one or more receptacles are eachconfigured to receive a complementary container such as a reagent tube(not shown) inserted from the upper side 512 of the connecting member.The lanes of the rack described herein are designed to have sufficientdepth and width to accommodate the various reagent tubes, receptacles,process tube, and pipette sheath of a given reagent holder, and toposition the process tube in communication with a heater/separator unit.

In FIG. 2B, a view of underside 514 is shown, having various struts 597connecting a rim of the connecting member with variously the sockets,process tube, and reagent tubes. Struts 597 are optional, and may beomitted all or in part, or may be substituted by, in all or in part,other supporting pieces that connect various parts of the holder to oneanother.

The one or more receptacles 550 are configured to accept reagent tubesthat contain, respectively, sufficient quantities of one or morereagents typically in solid form, such as in lyophilized form, forcarrying out extraction of nucleic acids from a sample that isassociated with the holder. The receptacles can be all of the same sizeand shape, or may be of different sizes and shapes from one another.Receptacles 550 are shown as having open bottoms, but are not limited tosuch topologies, and may be closed other than the inlet 552 in the upperside of connecting member 510. Preferably the receptacles 550 areconfigured to accept commonly used containers in the field of laboratoryanalysis, or containers suitably configured for use with the holderherein.

In one embodiment, the containers 554 containing lyophilized reagents,disposed in the receptacles 550, are 0.3 ml tubes that have been furtherconfigured to have a star-shaped pattern on their respective bottominterior surfaces. This is so that when a fluid has been added to thelyophilized reagents (which are dry in the initial package), a pipettetip can be bottomed out in the tube and still be able to withdraw almostthe entire fluid from the tube. The design of the star-pattern isfurther described elsewhere in U.S. patent application Ser. No.12/______, filed on even date herewith, and entitled “Reagent Tube”, inthe name of Handique et al., which application is incorporated herein byreference.

The embodiment of a reagent holder 804 is shown configured with a wastechamber 560, having an inlet aperture 562 in the upper side of theconnecting member. Waste chamber 560 is optional and, in embodimentswhere it is present, is configured to receive spent liquid reagents. Inother embodiments, where it is not present, spent liquid reagents can betransferred to and disposed of at a location outside of the holder, suchas, for example, a sample tube that contained the original sample whosecontents are being analyzed.

The embodiment of a reagent holder 804 is shown having a pipette sheath570. This is an optional component of the holders described herein. Itmay be permanently or removably affixed to connecting member 510, or maybe formed, e.g., moulded, as a part of a single piece assembly for theholder. Pipette sheath 570 is typically configured to surround the atleast one socket and a tip and lower portion of a pipette tip when thepipette tip is stationed in the at least one socket. In someembodiments, the at least one socket comprises four sockets. In someembodiments the at least one socket comprises two, three, five, or sixsockets.

Pipette sheath 570 typically is configured to have a bottom 576 and awalled portion 578 disposed between the bottom and the connectingmember. Pipette sheath 570 may additionally and optionally have one ormore cut-out portions 572 in the wall 578, or in the bottom 576. Inembodiments of the reagent holder having a pipette sheath, a purpose ofthe sheath is to catch drips from used pipette tips, and thereby toprevent cross-sample contamination, from use of one holder to another ina similar location, and/or to any supporting rack in which the holder issituated. Typically, then, the bottom 576 is solid and bowl-shaped(concave) so that drips are retained within it. An embodiment having nopipette sheath, could utilize, e.g., a drip tray or a drainage outlet,suitably placed beneath pipette tips located in the one or more sockets,for the same purpose and located under or in the bottom of the rack, asdescribed herein.

Process tube 520 can also be a snap-in tube, rather than being part ofan integrated piece. Process tube 520 is typically used for variousmixing and reacting processes that occur during sample preparation. Forexample, cell lysis can occur in process tube 520, as can extraction ofnucleic acids, such as DNA or RNA of a patient, and DNA or RNA of apathogen. Process tube 520 is then advantageously positioned in alocation that minimizes, overall, pipette head moving operationsinvolved with transferring liquids to process tube 520. Process tube 520is also located in the holder in such a position that, when the holderis inserted in a rack as further described herein, the process tube isexposed and accessible to a heater and separator, as further describedherein.

Some of the reagents contained in the holder are provided as liquids,and others may be provided as solids. In some embodiments, a differenttype of container or tube is used to store liquids from those that storethe solids.

Reagent tubes 540 are typically configured to hold liquid reagents, oneper tube. For example, in reagent holder embodiment 804, three reagenttubes are shown, containing respectively wash buffer, release buffer,and neutralization buffer, each of which is used in a sample preparationprotocol.

The reagent holder embodiment 804 has a connecting member that isconfigured so that the at least one socket, the one or more receptacles,and the respective apertures of the process tube, and the two or morereagent tubes, are all arranged linearly with respect to one another(i.e., their midpoints lie on the same axis). However, the holdersherein are not limited to particular configurations of receptacles,process tube, sockets, reagent tubes, and waste chamber if present. Forexample, a holder may be made shorter, if some apertures are staggeredwith respect to one another and occupy ‘off-axis’ positions. The variousreceptacles, etc., also do not need to occupy positions with respect toone another that are the same as those shown in FIGS. 10A and 10B. Thus,in FIGS. 10A, and 10B, the process tube is on one end of the connectingmember, and the pipette sheath is at the other end, adjacent to, in aninterior position, a waste chamber and two or more reagent tubes. Stillother dispositions are possible, such as mounting the process tube onone end of the holder, mounting the process tube adjacent the pipettetips and pipette tip sheath (as further described herein), and mountingthe waste tube adjacent the process tube. It would be understood thatalternative configurations of the various parts of the holder give riseonly to variations of form and can be accommodated within othervariations of the apparatus as described, including but not limited toalternative instruction sets for a liquid dispensing pipette head,heater assembly, and magnetic separator, as further described herein.Each such configuration of the reagent holder can be accommodated by acorresponding variation in form of the rack described herein thatreceives one or more such holders.

The process tube also may have a low binding surface, and allowsmagnetic beads to slide up and down the inside wall easily withoutsticking to it. Moreover, it has a hydrophobic surface coating enablinglow stiction of fluid and hence low binding of nucleic acids and othermolecules.

In some embodiments, the holder comprises a registration member such asa mechanical key. Typically such a key is part of the connecting member510. A mechanical key ensures that the holder is accepted by acomplementary member in, for example, a supporting rack as describedherein or a receiving bay of an apparatus that controls pipettingoperations on reagents in the holder. Thus, embodiment 804 has amechanical key 592 that comprises a pair of rectangular-shaped cut-outson one end of the connecting member. This feature as shown additionallyprovides for a tab by which a user may gain a suitable purchase wheninserting and removing the holder into a rack or another apparatus.Embodiment 804 also has a mechanical key 590 at the other end ofconnecting member 510. Key 590 is an angled cutout that eases insertionof the holder into a rack, as well as ensures a good registrationtherein when abutting a complementary angled cut out in a recessed areaconfigured to receive the holder.

A reagent holder for use with a rack as described herein is typicallymade of a plastic such as polypropylene. The plastic is such that it hassome flexibility to facilitate placement into a rack, as furtherdescribed herein. The plastic is typically sufficiently rigid, however,so that the holder will not significantly sag or flex under its ownweight and will not easily deform during routine handling and transport,and thus will not permit reagents to leak out from it.

The holder is typically such that the connecting member, process tube,the two or more reagent tubes, and the waste chamber (if present) aremade from a single piece, made from a material such as polypropylene.

Liquid Dispenser

Additionally, the heater and separator described herein can beconfigured to operate in conjunction with liquid processing operations,such as carried out by an automated pipette head. An exemplary automatedpipette head is described in U.S. provisional application Ser. No.60/959,437, filed Jul. 13, 2008, and in U.S. patent application Ser. No.12/173,023, filed Jul. 14, 2008, entitled “Integrated Apparatus forPerforming Nucleic Acid Extraction and Diagnostic Testing on MultipleBiological Samples”, in the name of Williams, et al., all of which areincorporated herein by reference in their entirety. As reactions arecarried out in a process tube that, for example, is part of a reagentholder as described elsewhere herein, the heater is controllably heatedat various stages as desired and in concert with various pipettingoperations. Similarly, the magnetic separator is controllably broughtinto proximity with a process tube as required at various stages in aprocess.

Typical features of an automated pipette head suitable for operatingwith the heater and separator as described herein include at least: anability to pick up pipette tips from the one or more sockets in areagent holder, and to return pipette tips to such sockets after use; tostrip and discard a pipette tip from a pipette head after use or uponencountering an error; move a pipette tip with precision from onelocation of a given holder to another so that, for example, liquidreagents can be located and added to solid reagents to make upsolutions, and various liquid reagents can be mixed with one anotherduring a sample preparation protocol. Furthermore, it is desirable thatsuch an automated pipette device can operate on several, such as 2, 3,4, or 6, holders simultaneously when received by a rack, and therebyperform certain operations in parallel. Thus the pipette head shouldmove in three degrees of freedom.

EXAMPLES Example 1: Integrated Heater/Separator

In FIG. 7 an exemplary integrated magnetic separator and heater assemblyare shown. Magnetic separator 1400 and heater assembly 1401 werefabricated comprising twelve heat blocks aligned parallel to oneanother. Each heat block 1403 is made from aluminum, and has an L-shapedconfiguration having a U-shaped inlet for accepting a process chamber1402. Each heat block 1403 is secured and connected by a metal strip1408 and screws 1407. Magnet 1404 is a rectangular block Neodymium (orother permanent rare earth materials, K & J Magnetics, ForcefieldMagnetics) disposed behind each heat block 1403 and mounted on asupporting member. Gears 1406 communicate rotational energy from a motor(not shown) to cause the motorized shaft 1405 to raise and lower magnet1404 relative to each heat block. The motor is computer-controlled tomove the magnet at speeds of 1-20 mm/s. The device further comprises aprinted circuit board (PCB) 1409 configured to cause the heater assemblyto apply heat independently to each process chamber 1402 upon receipt ofappropriate instructions. In the exemplary embodiment, the device alsocomprises a temperature sensor and a power resistor in conjunction witheach heater block.

Example 2: Exemplary Chemistry Processes Performed by an AutomatedInstrument Sample Pre-Processing

For Urine Sample: Take 0.5 ml of urine and mix it with 0.5 ml ofcollection buffer. Filter the sample through a pre-filter (containingtwo membranes of 10 micron and 3 micron pore size). Place the sampletube in the position specified for the external sample tube in a12-holder rack.

For Plasma Sample: Take 0.5 ml of plasma and mix it with 0.5 ml ofcollection buffer. Place the sample tube in the position specified forthe external sample tube in the 12-holder rack.

For GBS swab samples: Take the swab sample and dip it in 1 ml ofcollection buffer. Place the sample tube in the position specified forthe external sample tube in the 12-holder rack.

The sample collection buffer contains 50 mM Tris pH 7, 1% Triton X-100,20 mM Citrate, 20 mM Borate, 100 mM EDTA, plus 1,000 copies of positivecontrol DNA.

Loading the Instrument and Starting Sample Processing

The following steps may be performed to initiate an analysis on samplesin batch.

-   -   1. Load PCR tube containing PCR master mix in one of the        specified snap-in location of the reagent holder.    -   2. Load PCR tube containing PCR probes and primers for the        target analyte under consideration in the specified location of        the reagent holder.    -   3. In case of two analyte test, load PCR tube containing probes        and primers for second analyte in the specified location of the        reagent holder.    -   4. Insert the reagent holder in a 12-holder rack in the same        lane as the sample tube under consideration.    -   5. Prepare and insert reagent holders for other samples in        consideration.    -   6. Load the 12-holder rack in one of the locations in the        instrument.    -   7. Load a 12-sample cartridge in the cartridge tray loading        position.    -   8. Start operation.

Liquid Processing Steps

The following steps may be performed to carry out sample preparation.

-   -   1. Using Pipette tip #1, the robot transfers the clinical sample        from the external sample tube to the process tube of the reagent        holder.    -   2. Using the same pipette tip, the robot takes about 100 μl of        sample, mixes the lyophilized enzyme and affinity beads,        transfers the reagents to the process tube. Mixing is performed        in the process tube by 5 suck and dispense operations.    -   3. The robot places pipette tip #1 at its designated location in        the reagent holder.    -   4. Heat the process tube to 60° C. and maintain it for 10        minutes.    -   5. After 5 minute of lysis, the robot picks up pipette tip #1        and mixes the contents by 3 suck and dispense operations.    -   6. The robot places pipette tip #1 at its designated location in        the reagent holder.    -   7. After 10 minutes of lysis, a magnet is moved up the side of        the process tube to a middle height of the sample and held at        that position for a minute to capture all the magnetic beads        against the wall the tube.    -   8. The magnet is brought down slowly to slide the captured beads        close to the bottom (but not the bottom) of the tube.    -   9. Using pipette tip #2, aspirate all the liquid and dump it        into the waste tube.    -   10. Aspirate a second time to remove as much liquid as possible        from the process tube.    -   11. Using the same pipette tip #2, withdraw 100 μl of wash        buffer and dispense it in the process tube. During this        dispense, the magnet is moved downwards, away from the process        tube.    -   12. Perform 15 mix steps to thoroughly mix the magnetic beads        with the wash buffer.    -   13. Wait for 30 seconds.    -   14. Move magnet up to capture the beads to the side and hold for        15 seconds.    -   15. Using pipette tip #2, aspirate wash buffer twice to remove        as much liquid as possible and dump it back in the wash tube.    -   16. Move magnet down away from the process tube.    -   17. Place pipette tip #2 in its specified location of the        reagent holder.    -   18. Pick up a new pipette tip (tip #3) and withdraw 8-10 μl of        release buffer and dispense it over the beads in the process        tube.    -   19. Wait for 1 minute and then perform 45 mixes.    -   20. Heat the release solution to 85° C. and maintain temperature        for 5 minutes.    -   21. Place pipette tip #3 in its specified location of the        reagent holder.    -   22. Bring magnet up the tube, capture all the beads against the        tube wall and move it up and away from the bottom of the tube.    -   23. Pick up a new pipette tip (tip #4) and withdraw all the        release buffer from the process tube and then withdraw 3-10 μl        of neutralization buffer, mix it in the pipette tip and dispense        it in the PCR tube. (In case of two analyte detections, dispense        half of the neutralized DNA solution into first PCR tube and the        rest of the solution in the second PCR tube.    -   24. Using pipette tip #4, mix the neutralized DNA with the        lyophilized reagents by 4-5 suck and dispense operations and        withdraw the entire solution in the pipette tip.    -   25. Using pipette tip #4, load 6 μl of the final PCR solution in        a lane of the 12-up cartridge.

Real-Time PCR

After all the appropriate PCR lanes of the PCR cartridge are loaded withfinal PCR solution, the tray containing the cartridge moves it in thePCR Analyzer. The cartridge is pressed by an optical detection read-headagainst the PCR heater. Heaters activate valves to close either ends ofthe PCR reactor and real-time thermocycling process starts. Aftercompleting appropriate PCR cycles (˜45 cycles), the analyzer decideswhether the sample has the target DNA based on the output fluorescencedata, and issues an indication of the same.

Example 3: Exemplary Heater/Separator

Heaters for each of 24 process tubes, such as for carrying out lysis,can be individually software controlled. The lysis ramp times (e.g., thetime that it takes for the water in a lysis tube to rise from atemperature of approximately 2.5° C. to a given temperature) can be lessthan 120 seconds for a rise to 50° C. and less than 300 seconds for arise to 75° C. The lysis temperature (e.g., as measured in the watercontained in a lysis tube) can be maintained, by the heaters, to within±3° C. of the desired temperature. The accessible lysis temperaturerange can be from about 40° C. to about 82° C. Each of the heaters maydraw about 16 Watts or more of power when in operation. The lysis heatercan be designed to maximize the thermal transfer to the process tube,and also accommodate the tolerances of the various parts. The heaterscan permit the tubes to be in direct contact with the magnets (describedin more detail herein). The heaters may be adjustable in the horizontalplane during assembly and typically do not interfere with the covers ofthe system when installed.

Magnets are also included in the system, and the heater and magnetrelated mechanisms fit beneath a rack that contains a number of reagentholders, and do not interfere with rack insertion or registration. Themagnets may be high-flux magnets (e.g., have about a 1,000 gauss, orgreater, flux as measured within a given process tube), and be able tomove a distance sufficient to achieve magnetic bead separation in one ormore of the lysis tubes filled to a volume of 900 μL. The magnets can besoftware-controllable at movement rates from about 1 mm/sec to about 25mm/sec. The wiring, included as part of the heater and controllerassemblies, can be contained and protected from potential spills (e.g.,spills of the process tubes). The magnets can be located about 1.25inches or greater from the bottom of the lysis tube when not in use andcan be retained in such a manner as to maximize contact with the lysistube while also preventing jamming.

The foregoing description is intended to illustrate various aspects ofthe technology. It is not intended that the examples presented hereinlimit the scope of the technology. The technology now being fullydescribed, it will be apparent to one of ordinary skill in the art thatmany changes and modifications can be made thereto without departingfrom the spirit or scope of the appended claims.

1.-28. (canceled)
 29. A method of extracting nucleic acids using a preparatory apparatus, the method comprising: inserting a device in a rack, wherein the device comprises a process chamber; applying a magnetic force to the process chamber with a magnetic separator, wherein the process chamber contains magnetic particles bound to one or more biomolecules, wherein the magnetic separator comprises a magnet; applying heat to the process chamber with a heater assembly, wherein the heater assembly comprises a heater, wherein the magnet is positioned adjacent to a first side of the process chamber when the device is received in the rack, and wherein the heater is positioned adjacent to a second side of the process chamber when the device is received in the rack, wherein the first side is opposite the second side.
 30. The method of claim 29, wherein the magnet is moved and the process chamber is heated at the same time.
 31. The method of claim 29, further comprising sensing a temperature.
 32. The method of claim 29, further comprising moving the magnet from a first position to a second position relative to the process chamber.
 33. The method of claim 29, further comprising moving the magnet along a shaft.
 34. The method of claim 29, further comprising moving the magnet in close proximity to the process chamber.
 35. The method of claim 29, wherein the heater assembly is adjacent to at least two sides of the process chamber.
 36. The method of claim 29, wherein applying heat to the process chamber comprises raising the temperature of a sample in the process chamber from room temperature to about 65° C. in less than 3 minutes.
 37. The method of claim 29, wherein applying a magnetic force further comprises moving the magnetic particles against a wall of the process chamber.
 38. The method of claim 29, wherein applying a magnetic force further comprises concentrating the magnetic particles in a portion of the process chamber.
 39. The method of claim 29, wherein the magnetic particles are in suspension in solution in the process chamber, and wherein applying a magnetic force further comprises collecting the suspended magnetic particles into a location inside the process chamber.
 40. The method of claim 29, wherein the heater is aligned with the process chamber.
 41. The method of claim 29, further comprising moving the magnet along a fixed axis.
 42. The method of claim 41, wherein, during at least a portion of the motion, the magnet maintains close proximity to the process chamber.
 43. The method of claim 41, wherein the axis is vertical.
 44. The method of claim 29, further comprising separating the magnetic particles, while in solution.
 45. The method of claim 29, wherein the heater assembly and magnetic separator operate together.
 46. The method of claim 29, wherein the heater assembly and magnetic separator permit successive heating and separation operations to be performed on liquid materials in the process chamber.
 47. The method of claim 29, wherein the heater assembly and magnetic separator permit heating and separation operations without transporting either the liquid materials or the process chamber to a different location to perform either heating or separation.
 48. The method of claim 29, further comprising performing fluid transfer operations on fluids in the device using a liquid dispenser. 